Dynamic Combinatorial Optimization of In Vitro and In Vivo Heparin Antidotes

Heparin-like macromolecules are widely used in clinics as anticoagulant, antiviral, and anticancer drugs. However, the search of heparin antidotes based on small synthetic molecules to control blood coagulation still remains a challenging task due to the physicochemical properties of this anionic polysaccharide. Here, we use a dynamic combinatorial chemistry approach to optimize heparin binders with submicromolar affinity. The recognition of heparin by the most amplified members of the dynamic library has been studied with different experimental (SPR, fluorescence, NMR) and theoretical approaches, rendering a detailed interaction model. The enzymatic assays with selected library members confirm the correlation between the dynamic covalent screening and the in vitro heparin inhibition. Moreover, both ex vivo and in vivo blood coagulation assays with mice show that the optimized molecules are potent antidotes with potential use as heparin reversal drugs. Overall, these results underscore the power of dynamic combinatorial chemistry targeting complex and elusive biopolymers.

S-2 Dynamic combinatorial screening setup and results. S-4 Figure S1. Selected representative Ion-Selective UPLC traces for library members obtained from the reductive amination reaction in the absence (A) or in the presence (B) of heparin. The two numbers on the up-right corner of each chromatogram correspond to the m/z values and the peak area, respectively. In many cases, the reaction with Hep produced a simplified IS-UPLC trace, suggesting that in the absence of the template, several isomers corresponding to different alkylation sites were obtained. The identity of the amplified isomer was corroborated in each case by co-injection of the DCL with the pure molecules synthesized as a single isomer bearing the alkylation at the primary amine nitrogen atoms (see below).
S-5 Synthetic protocols and characterization data. Scheme S1. General synthesis of symmetric polyamine binders.
Spermine (1 eq) was dissolved in THFanh at 0°C. Aldehyde of interest R1 (2.2 eq) was then added dissolved in THFanh. The solution was stirred overnight. Then, NaBH3CN (4.4 eq) was added and the reaction was stirred 24h. After addition of H2O and HCl 1M (wait 1h after additions), THF was removed by rotavaporation. Reaction mixture was purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA) to yield 3R1R1 as pure product (Yield around 60% and purity 90-99% by HPLC).
Scheme S2. General synthesis of asymmetric polyamine binders.
Step 1: Spermine (1eq) was dissolved in anhydrous THF at 0°C. First aldehyde of interest R1 (0.9 eq) dissolved in anhydrous THF was dropwise added and the reaction was stirred overnight. The day after, NaBH3CN (2 eq) was added and stirred 24h. Reaction was stopped by addition of H2O and 1M HCl (1h after additions). THF was carefully evaporated in vacuum and reaction crude was purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA). 3R1 were obtained as pure product (yield around 30% and purity 90-99% by HPLC). Some 3R1R1 was also isolated.
Step 2: 3R1 (1eq) was dissolved in 5mL of Methanol at 0°C. Second aldehyde of interest R2 (1.2 eq) was added dissolved in MeOH. The day after, NaBH3CN (2 eq) was added and the reaction stirred overnight. Reaction was stopped by addition of H2O. Solvent was removed and crude purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA). 3R1R2 was obtained as pure product (yield around 70% and purity 90-99% by HPLC).

Aggregation studies with 3FF
The aggregation trend of 3FF in aqueous media was studied using three experimental techniques: NMR, fluorescence spectroscopy and DLS. Different parameters were tested, such as pH, time, temperature and overall concentration of the samples.
Aggregation of 3FF by NMR. Figure S2. Effect of the overall concentration of 3FF: 1D 1 H NMR spectra of 3FF at two concentrations, 0.1 (Bottom) and 0.5 mM (Top). NMR samples prepared in aqueous buffer (5 mM Tris/50 mM NaCl pH* 7.2) from stored samples, so we already observe aggregation just after dilution from stock solution (prepared in DMSO-d6). (bottom) partial 1D 1 H NMR spectra at neutral pH at two times after sample preparation (from stored sample, pH* 7.2, 298K, 5 mM Tris-d11, 50 mM NaCl). The corresponding D values are displayed in Table S7.

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Aggregation of 3FF by fluorescence spectroscopy Figure S6. Effect of the concentration on the aggregation of 3FF: Fluorescence spectra (left) of 3FF in Tris buffer at pH 7.1 at different concentrations. The ratio between the excimer (422 nm) and monomer (364 nm) emission bands of the naphthol fluorophore (right) shows a complex dependence on the overall concentration of 3FF, which is an indication of aggregation in solution, especially when [3FF] > 4 M. Figure S7. Fluorescence emission spectra of 3FF (10 M) at different pH values. The spectra show a higher amount of the excimer when increasing the pH.

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Aggregation of 3FF by Dynamic Light Scattering (DLS). Figure S8. Autocorrelation function (left) and particle size distribution (right) obtained from three DLS experiments performed with 20 M solutions of 3FF in 3 mM BisTris buffer at pH 7.5. We repeated the DLS measurements after 48 hours of sample preparation and, despite we observed scattering, the autocorrelation function did not allow the accurate determination of the particle size. The obtained scattering suggests the presence of bigger particles after 48 hours, implying a further aggregation in solution at longer times.
We additionally carried out DLS measurements at either pH 4.0 or at lower at concentration of 3FF (1 M and 4 M). The scattering data showed the absence of detectable particles in these conditions, implying that aggregation of 3FF depends on its overall concentration and on the pH of the medium.

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Figure S18. Anomeric region of the overlayed 2D 1 H-1 H TOCSY spectra of 3.9 mM dp14 (brown) and upon addition of 0.5 mM 3AC (green). The dp14 chemical shift assignment was based on literature data (see Table S5).

Interaction between 3FF and different forms of Hep in solution
Interaction of 3FF with Hep by fluorescence spectroscopy Figure S21. Fluorescence emission spectra (left) of 3FF (10 M in 1 mM Bis-Tris buffer at pH 7.5) upon the addition of increasing amount of unfractioned heparin (concentration of Hep given in the inset). On the right, we show the plot of the ratio of the excimer/monomer emission bands versus the equivalents of Hep disaccharide repeating units. Since we keep constant the concentration of 3FF and the pH of the medium, the reduction of the relative emission of the excimer implies a strong 3FF-Hep interaction in solution. However, the inherent aggregation of 3FF precludes a quantitative analysis of the titration experiment.

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Interaction of 3FF with Hep by DLS Figure S22: Autocorrelation function (left) and particle size distribution (right) obtained from three DLS experiments performed with samples containing 20 M of 3FF and 6 mg/L of Hep (10 M in disaccharide repeating units) dissolved in 3 mM BisTris buffer at pH 7.5.
S-77 Figure S23. Autocorrelation function (left) and particle size distribution (right) obtained from three DLS experiments with the previous samples but after 48 hours.  Tables S3 and S6) suggesting a 3FF-Hep interaction that competes with the 3FF aggregation. Moreover, the apparent size of the species changes with time, implying a dynamic complex behavior of the supramolecular species in solution.

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Interaction of some selected binders with Hep by fluorescence spectroscopy Figure S29. Fluorescence emission spectra (left) of 3AF (10 M in 1 mM Bis-Tris buffer at pH 7.5) upon the addition of increasing amount of unfractioned heparin (concentration of Hep given in the inset). On the right, we show the plot of the excimer/monomer emission bands ratio versus the equivalents of heparin disaccharide repeating units. The titration suggests a complex binding mechanism implicating the 3AF aggregation in competition with the Hep interaction. Figure S30. Fluorescence emission spectra of either 3AG (left) or 3GG (right), each at 10 M in 1 mM Bis-Tris buffer at pH 7.5, upon the addition of increasing amount of unfractioned heparin (concentration of Hep given in the insets). The arrows indicate the evolution of the spectra upon titration. Both experiments support the Hep binding, although a complex process is evident most likely due to the co-existence of different species in solution including the aggregation of the binder. The higher intensity of the excimer band throughout the titration experiments suggests that binding is dominated by aggregation for these two binders.

Molecular modeling studies
The interaction between heparin and selected ligands, namely 3AC, 3AL and 3FF, was studied by molecular dynamics. Comercial heparin is a polydisperse mixture of fragments of different length, constituted by the repetition of dimers of L-iduronic acid and D-glucosamine which are sulfated to a varying degree. In addition, it has been shown that two conformers of the iduronic acid residue are present in aqueous solutions of heparin-like saccharides, namely 1 C4 and 2 SO. 2 Thus, although a systematic study on the influence of the heparin sequence and conformation on ligand binding goes beyond the scope of our work, we prepared four different heparin models formed by eight dimers that differed on their sequence, namely were always in the 4 C1 conformation, two different initial conformations, 1 C4 and 2 SO, for the eight IdoA[2S] pyranose rings were considered. Thus, 4 different models were generated that were named: ( 4 C1)Glc-( 1 C4)Ido, ( 1 C4)Ido-( 4 C1)Glc, ( 4 C1)Glc-( 2 SO)Ido, and ( 2 SO)Ido-( 4 C1)Glc.
Simulations were performed in which six molecules of ligand and one molecule of Hep dp16 in explicit solvent were allowed to interact. The results showed small differences between ligands and between heparin models ( Figure S34), indicating that essentially all ligand molecules could bind to the heparin molecules during most of the simulations, establishing around 5 H-bond interactions per ligand molecule and with most of the atoms of every ligand molecule within close distance of the heparin molecule. In contrast, in the competence simulations between 3AC/3FF and 3AC/3AL a clear decrease in the number of H-bonds and ligand atoms within short distance from heparin is observed for the three ligands ( Figures S35 and S36). However, the decrease is in most cases larger for 3FF and 3AL than for 3AC, suggesting that the 3AC molecules have a higher affinity for heparin, in agreement with our experimental results. The differences between binding to the different Hep models are small and difficult to analyze without performing more simulations, thus no conclusions can be drawn in this respect.